Functional Characterization of Flavanone 3-Hydroxylase (F3H) and Its Role in Anthocyanin and Flavonoid Biosynthesis in Mulberry

Mulberry (Morus spp., Moraceae) is an important economic crop plant and is rich in flavonoids and anthocyanidins in ripe fruits. Anthocyanins are glycosides of anthocyanidins. Flavanone 3-hydroxylase (F3H) catalyzes the conversion of naringenin into dihydroflavonols and is responsible for the biosynthesis of flavonols and anthocyanidins. In this study, MazsF3H was cloned and characterized from Morus atropurpurea var. Zhongshen 1. Conserved motif analysis based on alignment and phylogenetic analysis indicated that MazsF3H belonged to 2-oxoglutarate-dependent dioxygenase and MazsF3H clustered with F3Hs from other plants. MazsF3H was located in both nucleus and cytosol. MazsF3H was expressed in stems, leaves, stigmas and ovaries, except buds. F3H expression levels showed a positive and close relationship with anthocyanin content during the anthocyanin-rich fruit ripening process, while it showed a negative correlation with anthocyanin content in LvShenZi, whose fruits are white and would not experience anthocyanin accumulation during fruit ripening. Significantly different F3H expression levels were also found in different mulberry varieties that have quite different anthocyanin contents in ripe fruits. Overexpression MazsF3H in tobacco showed unexpected results, including decreased anthocyanin content. Down-regulation of F3H expression levels resulted in co-expression of the genes involved in anthocyanin biosynthesis and a significant decrease in anthocyanin content, but the change in total flavonoid content was subtle. Our results indicated that F3H may play quite different roles in different varieties that have quite different fruit colors. In addition, possible complex regulation of flavonoid biosynthesis should be further explored in some of the featured plant species.


Introduction
The flavonoid biosynthetic pathway has been well investigated and is thought to be a gold mine for metabolic engineering [1]. The application of biological engineering of flavonoids is prevalent, and there have been many studies on the modification of color, the change in medicinal plant composition, and the improvement of crop nutrition. The flavonoid class of secondary metabolites mainly contains anthocyanins, flavonols, flavones and proanthocyanidins [2,3]. Anthocyanin is essential to the physiological and biochemical activities of plants, including antioxidation, protection of plant damage, stress responses, and attracting animals to pollinate and spread seeds [4]. In recent years, more and more attention has been paid to anthocyanins because of their prominent role in medical resources [5].
The flavonoid pathway-related enzymes F3H, FLS and ANS all belong to the 2-oxoglutarate-dependent dioxygenase (2-ODD) subfamily [11,12]. F3H is a key enzyme in directing carbon flow towards the biosynthesis of 3-hydroxylated flavonoids and is responsible for the biosynthesis of flavonols and anthocyanidins [13]. Down-regulation of F3H in strawberry resulted in a great decrease in anthocyanin and a moderate decrease in flavonol content [14]. Overexpression of Lycium chinense LcF3H in tobacco showed increased content of flavan-3-ols and increased tolerance to drought stress [15]. However, overexpression of Sorghum bicolor SbF3H resulted in a subtle impact on flavonol production in tissues with endogenous F3H activities [13]. Carthamus tinctorius CtF3H showed diametrically opposite expression patterns in different phenotypes with orange-yellow flowers and white flowers when exposed to external methyl jasmonate (MeJA) treatment, which has been identified as an elicitor of flavonoid metabolites [16]. The above facts suggest that the function of F3H in flavonoid biosynthesis is highly complex, and that its impact on flavonoid profile might be species-specific or phenotype-specific.
Mulberry (Morus spp., Moraceae) plants are distributed widely in China and East Asia and are known as important economic plants with nutritional, medicinal, and ecological value [17,18]. Mulberry fruits are a nutritional foodstuff and are rich in flavonoids and anthocyanins. They are recommended as having antioxidant, antimicrobial, and antiinflammatory properties [18]. Previous studies have reported that expression levels of genes involved in anthocyanin biosynthesis, including CHS1, CHI, F3H1, F3 H1, and ANS showed a close correlation with anthocyanin content during the fruit ripening process [19,20]. Given the complex roles played by F3H in flavonoid and anthocyanin biosynthesis, it is still necessary to further elucidate how F3H functions in the flavonoid biosynthesis pathway and anthocyanin accumulation during fruit ripening in mulberry. In the present study, we functionally characterized F3H in mulberry and found its variety-specific impact on anthocyanin accumulation. The expression patterns of F3H differ greatly among different mulberry varieties during the fruit ripening process. Generally, down-regulation of F3H in mulberry can result in a decrease in anthocyanins and a subtle increase in flavonoid content, while overexpression of mulberry F3H in tobacco also resulted in a significant decrease in anthocyanins. This suggests that more complex regulation mechanisms of flavonoid profile partitioning may possibly exist in mulberry.

Molecular Cloning and Characterization of MazsF3H in Mulberry
An F3H gene with 1098 bp CDS (coding sequence) coding 365 amino acids, named MazsF3H, was successfully cloned in Morus atropurpurea var. Zhongshen 1. MazsF3H has been deposited in NCBI (Accession number: ON055162). The weight of the putative protein coded by MazsF3H is about 42 kDa. The alignment of MazsF3H with other functionally characterized F3Hs showed that high sequence identity (86.5% identity) could be observed among these F3Hs. Conserved motifs of 2-oxoglutarate-dependent dioxygenase, including the ferrous iron ligation motif HXDX55H and the 2oxoglutarate (2-ODD) binding motif RXS (RLS), were detected in MazsF3H ( Figure 1A). In addition, five commonly conserved motifs in plant 2-oxoglutarate-dependent dioxygenase were also indicated and were found in MazsF3H. Phylogenetic analysis of 2-oxoglutarate-dependent dioxygenases in plants showed that MazsF3H clustered with other F3Hs, including Vitis vinifera (Vv) F3H, Arabidopsis thaliana (At) F3H et al., and can be distinguished from FLSs and ANSs ( Figure 1B). Enzymatic assay of purified MazsF3H in vitro showed that MazsF3H can convert naringenins to dihydrokaempferols ( Figure 2).

Molecular Cloning and Characterization of MazsF3H in Mulberry
An F3H gene with 1098 bp CDS (coding sequence) coding 365 amino acids, named MazsF3H, was successfully cloned in Morus atropurpurea var. Zhongshen 1. MazsF3H has been deposited in NCBI (Accession number: ON055162). The weight of the putative protein coded by MazsF3H is about 42 kDa. The alignment of MazsF3H with other functionally characterized F3Hs showed that high sequence identity (86.5% identity) could be observed among these F3Hs. Conserved motifs of 2-oxoglutarate-dependent dioxygenase, including the ferrous iron ligation motif HXDX55H and the 2oxoglutarate (2-ODD) binding motif RXS (RLS), were detected in MazsF3H ( Figure 1A). In addition, five commonly conserved motifs in plant 2-oxoglutarate-dependent dioxygenase were also indicated and were found in MazsF3H. Phylogenetic analysis of 2-oxoglutarate-dependent dioxygenases in plants showed that MazsF3H clustered with other F3Hs, including Vitis vinifera (Vv) F3H, Arabidopsis thaliana (At) F3H et al., and can be distinguished from FLSs and ANSs ( Figure 1B). Enzymatic assay of purified MazsF3H in vitro showed that MazsF3H can convert naringenins to dihydrokaempferols ( Figure 2). 2oxoglutarate (2-ODD) binding motif RXS (RLS). Five conserved motifs in plant 2-oxoglutaratedependent dioxygenase were also marked; (B) maximum-likelihood tree was constructed using JTT + G model and 1000 bootstrap replicates were selected for evaluate the reliability of tree, MazsF3H was indicated full triangle. All sequences used for alignment and phylogenetic analysis are listed in Table S1.
Molecules 2022, 27, x FOR PEER REVIEW Figure 1. Alignment and phylogenetic analysis of F3Hs: (A) alignment of mulberry MazsF3 other F3Hs from different plants. Red boxes indicate the ferrous iron ligation motif HXDX55 a 2oxoglutarate (2-ODD) binding motif RXS (RLS). Five conserved motifs in plant 2-oxoglu dependent dioxygenase were also marked; (B) maximum-likelihood tree was constructed usi + G model and 1000 bootstrap replicates were selected for evaluate the reliability of tree, M was indicated full triangle. All sequences used for alignment and phylogenetic analysis are li Table S1.

Subcellular Location of MazsF3H
Subcellular location of MazsF3H was indicated by yellow fluorescent protein and showed that MazsF3H was located in both the cytosol and nucleus ( Figure 3 cytosol and nucleus locations of structure genes involved in flavonoid biosynthesis way has been reported in several plants and is supposed to correspond with the biosynthesis of flavonoids in the cytosol and the nucleus [21,22].

Subcellular Location of MazsF3H
Subcellular location of MazsF3H was indicated by yellow fluorescent protein (YFP) and showed that MazsF3H was located in both the cytosol and nucleus ( Figure 3). The cytosol and nucleus locations of structure genes involved in flavonoid biosynthesis pathway has been reported in several plants and is supposed to correspond with the in situ biosynthesis of flavonoids in the cytosol and the nucleus [21,22]. Molecules 2022, 27, x FOR PEER REVIEW 5 of 13

Expression Profile of F3H and Its Relationship with the Accumulation of Anthocyanins
MazsF3H was expressed in all detected organs except buds and showed the highest expression level in leaves. Moderate expression levels were observed in stems, ovaries, and stigmata ( Figure 4A). To further study the relationship between F3H expression level and anthocyanin content during fruit ripening, both F3H expression levels and anthocyanin content of mulberry fruit at different growth stages were detected in two mulberry varieties: Zhongshen 1 (purple fruit) and LvShenZi (white fruit) ( Figure 4C). Quite different expression patterns of F3H were observed in Zhongshen1 and LvShenZi. In Zhongshen 1, which is rich in anthocyanins in ripe fruit, F3H showed increased expression level along with the ripening process and had a significant positive correlation with anthocyanin accumulation ( Figure 4B and Table 1). Meanwhile, in LvShenZi, in which there was no anthocyanin accumulation during fruit ripening, F3H showed a significant negative correlation with anthocyanin content during fruit ripening. Further study on F3H expression levels in ripe fruits from different mulberry varieties were also performed, and the results showed that F3H had obvious high expression levels in anthocyanin-rich fruit, but without significant correlation with anthocyanin accumulation ( Figure 4D and Table 1). The above results suggest that F3H may play different roles in mulberry varieties that have quite different anthocyanin contents, and that the regulation of anthocyanin content is more complex.

Expression Profile of F3H and Its Relationship with the Accumulation of Anthocyanins
MazsF3H was expressed in all detected organs except buds and showed the highest expression level in leaves. Moderate expression levels were observed in stems, ovaries, and stigmata ( Figure 4A). To further study the relationship between F3H expression level and anthocyanin content during fruit ripening, both F3H expression levels and anthocyanin content of mulberry fruit at different growth stages were detected in two mulberry varieties: Zhongshen 1 (purple fruit) and LvShenZi (white fruit) ( Figure 4C). Quite different expression patterns of F3H were observed in Zhongshen1 and LvShenZi. In Zhongshen 1, which is rich in anthocyanins in ripe fruit, F3H showed increased expression level along with the ripening process and had a significant positive correlation with anthocyanin accumulation ( Figure 4B and Table 1). Meanwhile, in LvShenZi, in which there was no anthocyanin accumulation during fruit ripening, F3H showed a significant negative correlation with anthocyanin content during fruit ripening. Further study on F3H expression levels in ripe fruits from different mulberry varieties were also performed, and the results showed that F3H had obvious high expression levels in anthocyanin-rich fruit, but without significant correlation with anthocyanin accumulation ( Figure 4D and Table 1). The above results suggest that F3H may play different roles in mulberry varieties that have quite different anthocyanin contents, and that the regulation of anthocyanin content is more complex.

Expression Profile of F3H and Its Relationship with the Accumulation of Anthocyanins
MazsF3H was expressed in all detected organs except buds and showed the highest expression level in leaves. Moderate expression levels were observed in stems, ovaries, and stigmata ( Figure 4A). To further study the relationship between F3H expression level and anthocyanin content during fruit ripening, both F3H expression levels and anthocyanin content of mulberry fruit at different growth stages were detected in two mulberry varieties: Zhongshen 1 (purple fruit) and LvShenZi (white fruit) ( Figure 4C). Quite different expression patterns of F3H were observed in Zhongshen1 and LvShenZi. In Zhongshen 1, which is rich in anthocyanins in ripe fruit, F3H showed increased expression level along with the ripening process and had a significant positive correlation with anthocyanin accumulation ( Figure 4B and Table 1). Meanwhile, in LvShenZi, in which there was no anthocyanin accumulation during fruit ripening, F3H showed a significant negative correlation with anthocyanin content during fruit ripening. Further study on F3H expression levels in ripe fruits from different mulberry varieties were also performed, and the results showed that F3H had obvious high expression levels in anthocyanin-rich fruit, but without significant correlation with anthocyanin accumulation ( Figure 4D and Table 1). The above results suggest that F3H may play different roles in mulberry varieties that have quite different anthocyanin contents, and that the regulation of anthocyanin content is more complex.   The significance is marked using * (0.01 < p < 0.05), ** (0.001 < p < 0.01).

Overexpression of MazsF3H in Tobacco Resulting in Change in Anthocyanin Content
Transient overexpression of MazsF3H in tobacco was performed, and three transgenic tobacco lines OE-F3H#1, #2, #3 were obtained and confirmed by qRT-PCR, indicating the significant overexpression of MazsF3H ( Figure 5A). However, the anthocyanin content in the overexpression tobacco leaves showed a significant decrease, while the total flavonoids showed no significant change except in OE-F3H#2, which had decreased flavonoid content ( Figure 5B,C).

Overexpression of MazsF3H in Tobacco Resulting in Change in Anthocyanin Content
Transient overexpression of MazsF3H in tobacco was performed, and three transgenic tobacco lines OE-F3H#1, #2, #3 were obtained and confirmed by qRT-PCR, indicating the significant overexpression of MazsF3H ( Figure 5A). However, the anthocyanin content in the overexpression tobacco leaves showed a significant decrease, while the total flavonoids showed no significant change except in OE-F3H#2, which had decreased flavonoid content ( Figure 5B,C).

Down-Regulation of F3H Resulting in Decreased Anthocyanin Content and Increased Total Flavonoid Content in Mulberry
qRT-PCR for F3H was performed to determine the knock-down efficiency of each positive transgenic mulberry. Expression levels of F3H decreased by about 61.5-75.2% in transgenic mulberry plants injected with pTRV2-MazsF3H compared to that in CK ( Figure  6A). The anthocyanin and total flavonoid contents exhibited no significant change in VIGS-F3H#1, which suffered a 61.5% decrease in F3H expression level. The anthocyanin contents were significantly decreased in VIGS-F3H#2 (75.2% decrease in F3H expression level) and VIGS-F3H#3 (70.1% decrease in F3H expression level) ( Figure 6B,C). This suggests that severe decrease in F3H expression level can result in a significant decrease in anthocyanins.

Down-Regulation of F3H Resulting in Decreased Anthocyanin Content and Increased Total Flavonoid Content in Mulberry
qRT-PCR for F3H was performed to determine the knock-down efficiency of each positive transgenic mulberry. Expression levels of F3H decreased by about 61.5-75.2% in transgenic mulberry plants injected with pTRV2-MazsF3H compared to that in CK ( Figure 6A). The anthocyanin and total flavonoid contents exhibited no significant change in VIGS-F3H#1, which suffered a 61.5% decrease in F3H expression level. The anthocyanin contents were significantly decreased in VIGS-F3H#2 (75.2% decrease in F3H expression level) and VIGS-F3H#3 (70.1% decrease in F3H expression level) ( Figure 6B,C). This suggests that severe decrease in F3H expression level can result in a significant decrease in anthocyanins.
The expression changes in structure genes involved in the flavonoid pathway were also determined when F3H was knocked-down. The genes involved in flavonoid precursor biosynthesis, including CHS, CHI2 and CHIL (CHI-like, IV class CHI), showed quite different changes. CHS showed increased expression, while CHI2 showed decreased expression levels in mulberry plants, with >70% decrease in F3H expression, while there was no significant change in VIGS-F3H#1 ( Figure 6D,E). CHIL, which has been reported to be a CHS interaction protein and to have a role in enhancing flavonoid biosynthesis, showed no significant change ( Figure 6F). The genes involved in anthocyanin biosynthesis, including F3 H and ANS, were significantly decreased ( Figure 6G,I). FLS, which is responsible for flavonol biosynthesis, showed significantly increased expression levels in F3H downregulated mulberry ( Figure 6H). The above results show that down-stream anthocyanin biosynthesis-related genes F3 H and ANS are co-expressed with F3H and could be responsible for anthocyanin accumulation, while genes involved in the biosynthesis of different flavonoid profiles have different responses to the disturbance of F3H expression levels. The expression changes in structure genes involved in the flavonoid pathway were also determined when F3H was knocked-down. The genes involved in flavonoid precursor biosynthesis, including CHS, CHI2 and CHIL (CHI-like, IV class CHI), showed quite different changes. CHS showed increased expression, while CHI2 showed decreased expression levels in mulberry plants, with >70% decrease in F3H expression, while there was no significant change in VIGS-F3H#1 ( Figure 6D,E). CHIL, which has been reported to be a CHS interaction protein and to have a role in enhancing flavonoid biosynthesis, showed no significant change ( Figure 6F). The genes involved in anthocyanin biosynthesis, including F3′H and ANS, were significantly decreased ( Figure 6G,I). FLS, which is responsible for flavonol biosynthesis, showed significantly increased expression levels in F3H downregulated mulberry ( Figure 6H). The above results show that down-stream anthocyanin biosynthesis-related genes F3′H and ANS are co-expressed with F3H and could be responsible for anthocyanin accumulation, while genes involved in the biosynthesis of different flavonoid profiles have different responses to the disturbance of F3H expression levels.

Discussion
Many previous studies have reported the relationships between structure genes involved in flavonoid biosynthesis and the accumulation of flavonoids and anthocyanins

Discussion
Many previous studies have reported the relationships between structure genes involved in flavonoid biosynthesis and the accumulation of flavonoids and anthocyanins [19,23,24]. In mulberry, CHS1, CHI, F3H1, F3 H1 (flavonoid 3 -hydroxylase), and ANS have been reported to correlate with anthocyanin biosynthesis during the fruit ripening process [19]. Our previous study on MmCHI1 and MmCHI2 from Morus multicaulis also showed that the dominant CHI MaCHI2 had a positive correlation with anthocyanin accumulation in fruit [25]. The present study shows that F3H expression levels have a positive correlation with anthocyanin accumulation in anthocyanin-rich mulberry fruits, while a negative correlation was found with anthocyanin content in mulberry fruits that did not require anthocyanin accumulation during fruit ripening process. These results indicate that different mechanisms may exist for regulation of flavonoid or anthocyanin biosynthesis in different mulberry varieties.
The subcellular locations of enzymes involved in secondary metabolite biosynthesis affect the utility of intermediate products and the efficiency of biosynthesis. Enzymes involved in the biosynthesis of secondary metabolites can form weakly bound, ordered complexes, which are referred to as "metabolons" [26]. Lignins and flavonoids are products of the phenylpropanoid pathway, and both lignin and flavonoid metabolons have been reported in many plants [26,27]. Flavonoid metabolons appear to be organized as multi-enzyme complexes, mainly at the endoplasmic reticulum (ER) and exclusively in cytoplasm [21]. However, flavonoids have been reported to be distributed in different cell compartments, including the cytosol, vacuole, ER, chloroplast, nucleus, and small vesicles, as well as in extracellular space, in different types of cells [21,22]. Despite the transport of flavonoid in cells, in situ biosynthesis of flavonoids in the nucleus has also been reported. Subcellular location analysis showed that some enzymes involved in flavonoid biosynthesis were located in both the nucleus and the cytosol. Arabidopsis AtCHS and AtCHI, CtF3H, Sm(eggplant)CHS, SmCHI, SmDFR, SmANS and MazsF3H, in this study, were all located in both the nucleus and the cytosol [21,28,29]. Meanwhile, lignin-related enzymes have been reported to be exclusively located in cytoplasm [30]. Given that flavonoids can bind to proteins and nucleic acids and play important roles in various biological processes, including as UV protectants and modifiers of auxin transport, in situ flavonoid biosynthesis in the nucleus may serve to protect DNA from UV or to control the transcription of genes [21,22,31]. It would be interesting to explore the biosynthesis mechanism of in situ flavonoid biosynthesis in the nucleus and its possible role in transcription regulation.
The regulation of structure genes involved in flavonoid biosynthesis could affect the accumulation of the products. Overexpression of CHS and CHI can enhance flavonoid and anthocyanin biosynthesis, and down-regulation of CHI results in a decrease in anthocyanin content [8,25,32]. The regulation of F3H expression may have quite different results in different plants. In addition, F3H even exhibited contrastings responses to methyl jasmonate (MeJA) in different plant phenotypes [16]. Our results also demonstrated the complex roles of F3H in flavonoid biosynthesis in different mulberry varieties. The expression of late anthocyanin biosynthesis genes (UFGT, ANS, DFR and CYP75B1) in fruits was much lower in non-pigmented mulberry, while the ratio of flavonols to flavonoids was higher in non-pigmented mulberry, which indicated the redirected the flux in the flavonoid pathway in non-pigmented mulberry [33]. The relatively higher expression levels of F3H in S1-S4 could be responsible for the accumulation of flavonols and the precursors of anthocyanins. During S5-S8, feedback regulation of F3H may result in decreased F3H expression levels while the accumulation of anthocyanins is still being slowly catalyzed by late anthocyanin biosynthesis enzymes, resulting in the observation of a negative correlation betwen F3H expression level and anthocyanin content. It is necessary to consider the phenotype first when trying to modify F3H to regulate flavonoid or anthocyanin biosynthesis. In addition, the multi-level regulation of flavonoid biosynthesis in plants has also been reported, including plant hormones, transcript factors and structure genes, and protein complex. Our previous study suggested that more complex metabolons may exist in mulberry [25]. Flavonoid biosynthesis in plants may be more complex, and more studies still need to be performed to reveal the regulation network covering different levels, especially in some featured plants.

Plant Materials
Samples of leaves, buds, stems, stigmata and ovaries, and fruits from Zhongshen 1 were collected in March 2021. These samples were used for gene cloning and expression profile analysis. The samples of mulberry fruits at different development stages from Zhongshen 1 and LvShenZi were reported in our previous study [25]. Morus atropurpurea variety Zhongshen 1(Mazs) has ripe fruits that are purple and Morus alba var. LvShenZi (LSZ) has ripe fruits that are white. All of the above samples were collected from the national mulberry germplasm field of the Chinese Academy of Agricultural Sciences, Zhenjiang and immediately stored at −80 • C. At least six mulberry fruits were collected from each mulberry plant. Two-week-old seedlings of Morus alba var. Fengchi were used to perform virus-induced gene silencing to down-regulation of F3H.

Isolation of RNA and cDNA Synthesis
Samples were ground with liquid nitrogen and total RNA was extracted using Plant RN38 Kit (Aidlab, Beijing, China) according to the manual. cDNA was synthesized with the PC54-TRUEscript RT kit (Aidlab, Beijing, China) according to the manufacturer's protocol.

Cloning of MazsF3H
The F3H coding region sequence was extracted from the morus alba genome using the annotation file based on the Blast results with AtF3H as query. The primers were designed and then synthesized using Sangon Biotech (Shanghai, China). The standard three-step PCR process was adopted with an annealing temperature of 54 • C to amplify F3H from Zhongshen 1. The PCR products were purified using SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China) and then cloned into the pMD18-T vector (Takara, Dalian, China). The sequences were deposited at GenBank (Accession number: ON055162) and named MazsF3H. Primer information is available in Table S2.

Alignment and Phylogenetic Analysis of F3Hs in Plants
MazsF3H and F3Hs from different plants, including Arabidopsis thialana AtF3H, Gynura bicolor GbF3H, Garcinia mangostana GmF3H, Gossypium hirsutum GhF3H and Vitis vinifera VvF3H, were aligned using DNAman 8.0 (Lynnon BioSoft, QC, Canada) with the default parameters to detect the conserved motifs. In addition, alignment of 2-oxoglutaratedependent dioxygenases (2-ODDs), including F3Hs, FLSs and ANSs, was performed to construct the phylogenetic tree. The phylogenetic tree was constructed using Mega 7.0 by means of the maximum-likelihood method using JTT substitution model and the G + I rates among sites model. The tree was assessed using the bootstrapping method with 1000 bootstrap replicates, and marked above nodes only if greater than 50 [34].

Prokaryotic Expression and Purification of MazsF3H
Recombinant plasmids pET28a-MazsF3H were constructed using seamless cloning (CV1901 kit, Aidlab, Beijing, China) and then confirmed by sequencing. Recombinant plasmids were transferred into E.coli BL21(DE3) cells and then incubated at 37 • C in LB media containing kanamycin (50 µg/mL). Finally, a concentration of 0.4 mM isopropy-β-D-thiogalactoside (IPTG) was added at OD = 0.6. Cells were collected by centrifugation at 4000 g after 4 h of incubation at 28 • C. The fusion protein was purified using Ni-NTA Sefinose Resin according to the user's manual (BBI Life Sciences, Shanghai, China). SDS-PAGE and Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China) were used to detect purified proteins and to determine the concentration of purified proteins.

Enzymatic Assay of MazsF3H
In vitro MazsF3H activity was determined as described by Si et al. (2022) with some modifications. The total 400 µL reaction mixture contained 25 mM Tris-HCl (pH 7.4), 0.4 mM naringenin, 0.5 mM dithiothreitol, 1 mM ascorbate acid, 0.2 mM ferrous sulfate, 1 mM 2-oxo-glutarate, and 10 µg or 20 µg recombinant proteins. Sample with protein that had been incubated in boiling water for 10 min were used as the negative control.

Expression Profile of F3H in Mulberry
qRT-PCR (quantitative real-time PCR) was performed to explore the expression of MazsF3H in different organs, its expression pattern throughout the fruit ripening process, and in the differences in its expression in ripe fruits of different mulberry varieties using an ABI StepOnePlus™ Real-Time PCR System (Foster City, CA, USA). The primers are presented in Table S2. Actin was used as a reference gene [35]. GraphPad Prism 8.0 was used to visualize the qRT-PCR results and perform ANOVA. p < 0.05 was denoted as significance. Firstly, three biological replicates were mixed, and then three technical replicates were performed, respectively, for qRT-PCR.

Subcellular Location of MazsF3H
The method used for the subcellular location of MazsF3H was the same as that reported in our previous study [36,37]. The pBI121 vector with MazsF3H gene and yellow fluorescent protein (YFP) fusion expression was constructed. The confirmed recombinant plasmids were transferred into Agrobacterium tumefaciens strain GV3101, which were then transferred into tobacco leaves via Agrobacterium-mediated transient transformation [38]. The YFP fluorescence in leaves was observed using a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Measurement of Anthocyanin and Total Flavonoid Content
The extraction and measurement of anthocyanin content were performed according to our reported methods, modified from the methods provided by Mehrtens et al. (2005) [39]. The anthocyanin content is given in cyanidin-3-glucoside equivalents. The total flavonoid content was measured with an aluminum nitrate method, using rutin as a reference substance according to a previous study [40].

Transient Overexpression of MazsF3H in Nicotiana Benthamiana
The recombinant plasmids pNC-1304-35S:MazsF3H were constructed using nimble cloning [41]. Both recombinant plasmids pNC-1304-35S:F3H and empty vector pNC-1304-35S:GFP, as the negative control, were transformed into Agrobacterium tumefaciens GV3101 and then transferred into N. benthamiana leaves via Agrobacterium-mediated transient transformation, as per a previous report [38]. Overexpression of MazsF3H was determined using qRT-PCR by comparing the expression levels of target genes in transgenic plants with those in the negative controls.

Obtaining MazsF3H VIGS Transgenic Mulberry
Virus-induced gene silencing (VIGS) was used to obtain MazsF3H down-regulated transgenic mulberry, in accordance with our previous report [25,42]. Agrobacterium tumefaciens containing recombinant plasmids pTRV2-MazsF3H, pTRV1 and pTRV2 (negative control) was cultured in transient transformation buffer and then transferred into mulberry leaves by means of pressure injection. Three independent mulberry plants were injected. The knock-down efficiency for the target genes was determined by qRT-PCR 15 days after injection by comparing the transgenic plants with the negative controls and wild types. The correlation analysis and ANOVA analysis were performed using SPSS19.0.

Conclusions
In conclusion, our results characterized MazsF3H from the Morus atropurpurea variety Zhongshen 1 and revealed that F3H may play quite different roles in different varieties that have quite different fruit phenotypes. Transcriptional regulation of F3H can result in a quite different impact on the genes involved in flavonoid precursor biosynthesis and the genes involved in anthocyanin biosynthesis, as well as unexpected changes in flavonoid or anthocyanin content. This potential complex regulation of flavonoid biosynthesis should be further explored in some of the featured plant species.